Methods of inducing ovulation

ABSTRACT

The present invention relates to methods of inducing ovulation in a female host comprising the administration of a non-polypeptide cyclic adenosine monophosphate (cAMP) level modulator to the female host. In another aspect, the invention provides for specific administration of the phosphodiesterase inhibitor prior to the luteal phase of the host&#39;s ovulatory cycle. Preferred non-polypeptide cAMP level modulator include phosphodiesterase inhibitors, particularly inhibitors of phosphodiesterase 4 isoforms.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S.application Ser. No. 09/928,268 filed Aug. 10, 2001, which claims thebenefit of U.S. provisional application No. 60/224,962, filed Aug. 11,2000, incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of enhancing fertilityin a female host by inducing ovulation with the administration of anon-polypeptide modulator of cAMP levels.

BACKGROUND OF THE INVENTION

[0003] Ovulation is the process where an ovum or ova are released fromthe ovaries. The timing of ovulation within the menstrual cycle is offoremost importance for fertilization. It is well recognized thatfollicles acquire the ability to ovulate following growth and maturationstimulated by the pituitary gonadotropins. Follicle stimulating hormone(FSH) is predominantly responsible for follicular growth and luteinizinghormone (LH) stimulates ovulation. This coordinated process ofgonadotropin-stimulated maturation of the follicle ensures delivery of acompetent ova at ovulation. The adequately prepared ovum is thenavailable for fertilization by sperm within hours after ovulation.

[0004] Ovulation is a finely timed process that is driven by pituitarygonadotropin stimulation of the ovary, and modified by the growth andbiochemical (e.g., steroidogenic, inhibin secretion, etc.) response offollicles to the gonadotropin stimulation. During the normal menstrualcycle in women these hormones exhibit cyclic patterns. The menstrualcycle can be functionally divided into three phases: the follicular, theovulatory and luteal phases. The follicular period begins at the end ofthe luteal phase of the preceding non-conceptive cycle, prior to orcoincident with the onset of menses. The cycle starts with a transientrise in blood levels of FSH that stimulates development of a cohort ofovarian follicles. The size of the follicles recruited to grow is about5 mm in diameter. In a natural menstrual cycle, usually one large ordominant follicle is established during the follicular phase, and it iscommitted to growth to maturation. In humans, the size of the folliclethat is considered ready to ovulate is about 15 mm or more in diameter.

[0005] The second critical event that occurs in the ovary during thefollicular phase is that granulosa cells within the ovarian folliclesacquire receptors for LH and become increasingly responsive to LH.Secretion of estradiol and estrone from the ovary increases slowly atfirst, in parallel to the increasing diameter of the follicle andsensitivity of the follicle to LH. The relatively rising levels ofestrogen and inhibin cause inhibition of gonadotropin releasing hormone(GnRH) secretion from the hypothalamus and gonadotropin secretion fromthe pituitary. Estrogen production reaches a maximum on the day beforethe LH peak and the neuroendocrine response to increased estrogen andgradually increasing concentrations of progesterone is the preovulatoryrelease of gonadotropins which is discussed below.

[0006] During the ovulatory phase there is a change in theneuroendocrine response to estrogen and progesterone. At this point inthe cycle, elevated estrogen elicits a preovulatory surge in serum FSHand LH levels, due to a positive feedback on the hypothalamus, estrogennow stimulating a surge in the levels of GnRH and subsequently FSH andLH release from the pituitary. This surge of gonadotropins induces thecompletion of follicular maturation and causes rupture of the dominantor Graafian follicle and discharge of the ovum some 16 to 24 hours afterthe LH peak. During the period following the preovulatory surge, serumestradiol levels temporarily decline and plasma progesterone levelsbegin to rise.

[0007] Following ovulation, the post-ovulatory ovarian follicle cellsunder the influence of LH are luteinized to form a corpus luteum—theluteal phase. The diagnostic markers of the luteal phase of themenstrual cycle are the marked increase in progesterone secretion by thecorpus luteum, and the uterine transformation that occurs in response toprogesterone. Associated with luteal progesterone production, there is aless pronounced increase in serum estrogen levels. As progesterone andestrogens increase, LH and FSH decline throughout most of the lutealphase. Towards the end of the luteal phase, in a non-conceptivemenstrual cycle, the corpus luteum regresses and serum FSH levels beginto rise to initiate follicular growth for the next cycle.

[0008] FSH and LH are distinguished from each other by their ability tostimulate follicular development or ovulation, respectively. Both agentsare known to stimulate an increase in intracellular cAMP concentrations.Agents that mimic cAMP such as forskolin or stable analogs of cAMP havebeen shown, in vitro, to resemble the effects of FSH in granulosa cellsfrom immature follicles, and to resemble the effects of LH in cells frommature follicles. Although alternative intracellular pathways have beenproposed for both FSH and LH, it is well accepted that cAMP isstimulated in response to both gonadotropins. If and when elevations incAMP levels are associated with follicular development and maturation orovulation induction depends on the cell types and the presence orabsence of the respective receptors. Indeed, it has been demonstratedthat mice which are deficient in a particular phosphodiesterase haveimpaired ovulation and diminished sensitivity of granulosa cells togonadotropins.

[0009] Infertility treatments currently in clinical use incorporate someof the regulatory events described above. One agent which stimulatesfollicular growth and is used for treatment of anovulation isclomiphene. Clomiphene is a nonsteroidal antiestrogen that competes forestrogens at their binding sites. It is thought that clomiphene binds toestrogen receptors in the hypothalamus and pituitary and blocks thenegative feedback exerted by ovarian estrogens. The result is increasedoutput of gonadotropins (FSH and LH) during the early part of thefollicular phase. The effect of clomiphene is to increase endogenous FSHserum levels and to improve the growth and maturation of follicles.Subsequently either endogenous LH or exogenous LH/CG induce ovulation inthese patients.

[0010] In addition to clomiphene, women have been treated with ovulationinduction regimens which include commercial preparations of the humangonadotropins, including follicle stimulating hormone (FSH) andluteinizing hormone (LH) or chorionic gonadotropin (CG), all of whichwere first obtained by purification of urine from pregnant women andmore recently by recombinant technology. In general, this treatment ishighly effective in stimulating folliculogenesis and steroidogenesis.Complications of this treatment result from the fact that thesepreparations and regimens can over-stimulate follicular development andmaturation of follicles. In a subset of patients, the ovary can becomehyperstimulated, which may result in multiple ovulations and,consequently, multiple births. Not only can ovarian hyperstimulation belife threatening to the mother, it also typically results in newbornswith lower birth weight, who subsequently require intensive care. It isbelieved that the principal complications attributed togonadotropin-induced hyperstimulation and multiple pregnancies probablyresult from the prolonged effects of hCG. In addition, use ofgonadotropins in ovulation induction regimens can result in injectionsite reactions, both local and systemic. Consequently, the developmentof ovulation induction regimens using orally active agents with mildergonadotropin-like activity as opposed to therapies that use potentinjectables would be of substantial benefit. More importantly, it wouldbe a significant advantage if ovulation induction regimens could bedeveloped which result in less ovarian hyperstimulation and,consequently, present less danger to the mother and produce healthiernewborns.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention provides a use of a non-peptidecAMP level modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the manufacture of a medicament for the induction ofovulation in a patient.

[0012] In a second aspect, the invention provides a use of a non-peptidecAMP level modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the induction of ovulation in a patient.

[0013] In a third aspect, the invention provides a use of a non-peptidecAMP level modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the manufacture of a medicament for the induction ofovulation in a patient, in a regimen whereby follicular maturation isinduced with clomiphene or an aromatase inhibitor, preferably anaromatase inhibitor selected from YM-511, Letrozole, Fadrozole, andAnastrozole, more preferably selected from Letrozole and Anastrozole,prior to ovulation induction.

[0014] In a fourth aspect, the invention provides a use of a non-peptidecAMP level modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the induction of ovulation in a patient, in a regimenwhereby follicular maturation is induced with clomiphene or an aromataseinhibitor, preferably an aromatase inhibitor selected from YM-511,Letrozole, Fadrozole, and Anastrozole, more preferably selected fromLetrozole and Anastrozole, prior to ovulation induction.

[0015] In a fifth aspect, the invention provides a kit for use ininducing ovulation, the kit comprising an ovulation inducing dose of anon-peptide cAMP level modulator, preferably a PDE inhibitor, morepreferably a PDE4 inhibitor, and instructions for its use in inducingovulation.

[0016] In a sixth aspect, the invention provides a kit for use inovulation induction and/or assisted reproductive technologies (ART), thekit comprising sufficient daily doses of FSH and/or a compound havingFSH effect, preferably clomiphene or an aromatase inhibitor, morepreferably an aromatase inhibitor selected from YM-511, Letrozole,Fadrozole, and Anastrozole, to cause follicular maturation, and anovulation inducing dose of a non-peptide cAMP level modulator,preferably a PDE inhibitor, more preferably a PDE4 inhibitor, andinstructions for the use of the kit in ovulation induction or ART.

[0017] In a seventh aspect, the invention provides a use of anon-peptide cAMP level modulator, preferably a PDE inhibitor, morepreferably a PDE4 inhibitor, for inducing ovulation in a patient, in aregimen wherein hCG or LH are also administered to induce ovulation, andthe hCG or LH are administered at a reduced dose compared to the amountof hCG or LH normally required to induce ovulation in the same patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a schematic representation of a generalized ovulationinduction regimen in humans.

[0019]FIG. 2 is a bar graph representation of the effect ofphosphodiesterase inhibitors in vitro (i.e., Compound 1 and Compound 2)on increasing intracellular cAMP levels in granulosa cells.

[0020]FIG. 3 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on follicle maturation inrats, in vivo.

[0021]FIG. 4 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on ovulation with CG inrats, in vivo.

[0022]FIG. 5 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on ovulation with andwithout CG in rats, in vivo.

[0023]FIG. 6 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 2) on ovulation with andwithout CG in rats, in vivo.

[0024]FIG. 7 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 2) on ovulation followingoral and subcutaneous administration.

[0025]FIG. 8 is a bar graph representation of the effect of PDEinhibitor Compound 3 on ovulation in vivo following oral andsubcutaneous administration.

[0026]FIG. 9 is a bar graph representation of the effect of PDEinhibitor Compound 4 on ovulation in the presence or absence of asub-effective dose of hCG in vivo.

[0027]FIG. 10 is a bar graph representation of the effect of PDEinhibitor Compound 2 on ovulation and fertility.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to methods of improving fertilityin a female host comprising the administration of a non-polypeptidecyclic adenosine monophosphate (cAMP) level modulator to the femalehost. In another aspect, the invention provides for specificadministration of the non-polypeptide cAMP level modulator to induceovulation of the dominant mature follicle prior to the luteal phase ofan ovulatory cycle. Preferred non-polypeptide cAMP level modulatorsinclude phosphodiesterase inhibitors, particularly inhibitors ofphosphodiesterase 4 isoforms.

[0029] Although effects of PDE inhibitors on LH-stimulatedsteroidogenesis from granulosa cells, in vitro, have been reported, thepresent invention describes two novel findings. First, the PDEinhibitors fail to enhance FSH-stimulated follicular growth in vivo,despite the accepted role of cAMP in both FSH and LH cellular pathways.Moreover, evidence is presented that PDE inhibitors did enhancegonadotropin-stimulated steroidogenesis in vitro, which furtherexemplifies the novel activity of the PDE inhibitor on LH-dependentovulation. Second, the PDE inhibitors increased the ovulation rate, invivo, in the absence of added LH or hCG. Considering the oral activityof PDE inhibitors, this second finding provides the basis for the firstpotential injection-free regimen for ovulation induction, since the PDEinhibitors can be used in conjunction with existing regimens asdescribed below.

[0030] The invention also provides for the stimulation of folliculardevelopment prior to the administration of a non-polypeptide cAMP levelmodulator which comprises the administration of an agent which increasesFSH concentrations during the follicular phase of the host's ovulatorycycle. The objective of the invention in increasing FSH relates solelyto follicular development and maturation and not ovulation induction.Preferred agents include FSH, itself, clomiphene, selective estrogenreceptor modulators, aromatase inhibitors and selective modulators ofthe neuroendocrine regulation of FSH production.

[0031] In still another aspect, the invention provides for theco-administration of a non-polypeptide cAMP level modulator with LH orchorionic gonadotropin (CG) prior to the luteal phase of the femalehost's ovulatory cycle. Co-administration can occur sequentially orsimultaneously, as well as by the same or different modes of delivery(e.g., parenterally and or orally). In addition, the invention providesfor the use of lower concentrations of LH or CG administered to the hostthan concentrations that are used in current ovulation inductionregimens and thereby lowering the likelihood of ovarianhyperstimulation.

[0032] Additionally, the present invention provides for the use of anon-polypeptide cAMP level modulator as a therapeutic agent inreplacement of or to enhance the effect of hCG or LH in the collectionof oocytes for in vitro fertilization.

[0033] Thus, the invention provides for the use of a non-polypeptidecAMP level modulator as a small molecule therapeutic (e.g.,phosphodiesterase inhibitors) that is administered orally rather than byinjection, the required route of administration for proteins and themode of administration in current ovulation induction regimens. Oraladministration avoids the acute and systemic side effects associatedwith such injections. Foremost, the small molecule therapeutic iseffective in inducing ovulations and can be administered alone or withor without LH or CG and alternatively, in lower concentrations of LH orCG than are currently used, and thus, lessen the occurrence of ovarianhyperstimulation and its associated risks. Consequently, multiple birthsand life threatening complications for the mother and newborns can beaverted. In addition, the present invention provides for the opportunityof earlier diagnostic testing for pregnancy than current ovulationinduction regimens involving the use of CG.

[0034] The treatment methods of the invention will be useful fortreatment of infertility in humans, but also in other mammals (such ashorses and livestock, e.g., cattle, sheep, cows and the like) and otherspecies, such as piscine (i.e., fish) and avian (i.e., fowl).

I. Definitions

[0035] In general, the following words or phrases have the indicateddefinition when used in the description, examples and claims.

[0036] “Administration” refers to the delivery of a therapeutic agentinto a female host. In the context of the present invention, this wouldinclude the delivery of a non-polypeptide cAMP level modulator and/or anagent which increases FSH concentrations. This term is more fullydescribed under the section entitled, “Pharmaceutical Compositions”contained herein.

[0037] “Ovulation” for the purposes herein refers to the process wherean ovum or ova are released from the ovaries. As midcycle approaches,there is a dramatic rise in estrogen, followed by an LH and to a lesserextent an FSH surge. This triggers the dominant follicle to ovulate.Ovulation consists of rapid follicular enlargement followed byprotrusion of the follicle from the surface of the ovarian cortex.Ultimately, rupture of the follicle results in the extrusion of anoocyte-cumulus complex. The remnant of the dominant follicle thenreorganizes to become the corpus luteum.

[0038] “Anovulation” refers to a lack of ovulation.

[0039] “Non-Polypeptide cAMP Level Modulator” refers to compounds thatare not composed of amino acids in their entirety, irrespective ofglycosylation, and act, directly or indirectly, to increaseintracellular levels of cAMP. Such compounds can increase cAMP levels bystimulating cAMP synthesis or by inhibiting its degradation, or both.Examples of modulators which increase the synthesis of cAMP includeactivators of adenyl cyclase such as forskolin. Examples of modulatorsthat decrease cAMP degradation include inhibitors of phosphodiesterasessuch theophylline.

[0040] “Female Host” means an individual of female gender of a speciesto which agents are administered in accordance with the presentinvention. Humans other mammalians and other species such as fish andfowl are included by definition herein.

[0041] “Phosphodiesterase Inhibitor” refers to chemical compounds whichblock or inhibit phosphodiesterases (PDE's) whose action is toinactivate their cyclic nucleotide targets (i.e., cAMP and cGMP) byhydrolytic cleavage of the 3′-phosphodiester bond, resulting in passiveaccumulation of specific cyclic nucleotides. Inhibitors can benon-selective for all phosphodiesterase isoforms or selective forspecific isoforms. See compounds cited herein.

[0042] “Phosphodiesterase Isoforms” refers to a family of isozymes orisoforms responsible for the metabolism or degradation of theintracellular second messengers, cAMP and cGMP. Specific isoforms canhave highly selective cellular and subcellular localizations. Examplesof phosphodiesterase isoforms include PDE3 and PDE4.

[0043] “Follicular Phase” refers to the first part of the menstrualcycle and is characterized by a progressive increase in circulatinglevels of estradiol and inhibin B by the developing Graafian follicle.

[0044] “Prior to the Luteal Phase” refers to the period of the menstrualcycle before the shift from the estrogen dominated follicular phase tothe progesterone dominated luteal phase. Prior to the luteal phase, theestrogen levels are generally greater than or equal to 150pg/ml/follicle for a follicle of 16 mm in diameter and the follicle sizeis generally no less than 14 mm in diameter. These criteria are notabsolute and will vary from patient to patient. In the context of thepresent invention and in terms of the timing of administration of thenon-polypeptide cAMP level modulator, the non-polypeptide cAMP levelmodulator can be administered to a female host at the time point of anexisting ovulation induction regimen at which hCG or LH is normallyadministered to said host.

[0045] “Ovulatory Cycle” or “Menstrual Cycle” refer to a series ofcyclical events over a species-specific period of time includingfollicular growth and development, recruitment, selection, dominance,ovulation, and corpus luteum formation and demise. Functionally, thecycle can be divided into three phases, the follicular, the ovulatoryand the luteal phases. This cycle can also be referred to the menstrualcycle.

[0046] “Ovulation Induction” refers to the process wherein apolypeptide(s) and/or synthetic chemical is used to bring aboutovulation in female hosts who are otherwise anovulatory, resulting ininduction of follicular rupture and ovulation of fertilizable oocytes.Ovulation induction does not include the preceding events in time duringthe ovulatory cycle of follicular maturation and development.

[0047] “Ovarian Hyperstimulation” refers to pharmacological interventionof an ovulatory or anovulatory menstrual cycle. It entails maturation ofmultiple follicles resulting in codominance of numerous follicles andthe availability of multiple fertilizable oocytes.

[0048] “Follicle” refers to the fluid filled sac that surrounds theovum, the sac also containing granulosa cells.

[0049] “Follicular Development” refers to the progressive growth anddevelopment of ovarian follicles, particularly during the follicularphase of the ovulatory cycle and leading to the recruitment anddominance of a follicle destined to ovulate.

[0050] “Follicle Stimulating Hormone (FSH) and Isoforms” refers to ahormone released by the pituitary that stimulates the growth of ovarianfollicles and isoforms of FSH as described, for example, in U.S. Pat.No. 5,087,615, incorporated by reference herein.

[0051] “Selective Estrogen Receptor Modulators” refers to chemicalcompounds or polypeptides that act as an estrogen receptor antagonist atthe level of the hypothalamus and the pituitary gland, and as an agonistat the level of the uterus. Examples of such modulators can includetamoxifen, raloxifene, toremifene, clomiphene and droloxifene. ReferenceShughrue et al (1999) is hereby incorporated by reference.

[0052] “Aromatase Inhibitors” refer to chemical compounds orpolypeptides that block or inhibit the activity of aromatase which is anenzyme that converts androgens to estrogens. Examples of aromataseinhibitors include Letrozole, Anastrozole and Vorozole. ReferencesTurner et al 1, (2000) and Shetty et al (1997) are hereby incorporatedby reference.

[0053] “Related Steroidogenic Enzymes” refers to any enzyme that isinvolved with the catalysis of biochemical reactions leading to thesynthesis of estrogen and progesterone including 3-β-hydroxysteroiddehydrogenase and inhibitors of this enzyme include daidzein, genistein,biochanin A and formononetin. Reference Cooke (1996) is herebyincorporated by reference.

[0054] “Clomiphene” refers to2-[4-(2-chloro-1,2-diphenylethenyl)phenoxy]-N,N-diethylethanamine andits salts.

[0055] “Lutenizing Hormone” refers to a hormone released by thepituitary that serves the dual purpose of causing a dominant follicle torelease its egg and stimulating the corpus luteum to secreteprogesterone.

[0056] “Reduced concentrations” refers to lower concentrations of anadministered agent when compared to standard levels of administeredagents. In the context of the present invention, lower concentrations ofLH or CG are administered than are administered in existing ovulationinduction regimens.

[0057] “Existing Ovulation Induction Regimens” refers to current methodsof inducing ovulation including the use of clomiphene, gonadotropins(i.e., FSH, LH and CG) or a combination of these agents to promotefolliculogenesis and induced ovulation in anovulatory females. Theregimens are varied in terms of the timing, frequency and concentrationof the agents administered. This definition includes modifications ofcurrent regimens which still require the administration of hCG or LH atsome time point during the ovulation induction regimen. The followingtreatises on female infertility, stimulated folliculogenesis andovulation induction are incorporated by reference herein: Adashi et al(1996) and Shoham et al (1999).

[0058] “Chorionic Gonadotropin” refers to a glycoprotein hormone that isbiologically and immunologically similar to pituitary LH. In normalpregnancy, CG is produced by the placenta and can be used as adiagnostic marker of pregnancy by elevated levels in serumconcentration. The acronym hCG refers to human chorionic gonadotropin.

[0059] “Agent which Increases FSH Concentrations” refers to anycomposition of matter, protein or synthetic chemical, which whenadministered to a female host increases the serum level concentrationsof FSH, either directly or indirectly, by administering FSH itself, oran agent which stimulates its endogenous production or inhibits itsendogenous degradation. The definition of this phrase and agent includescompounds which may not increase FSH concentrations but have folliclestimulating hormone biological function and activity.

II. Principles of Ovulation Induction

[0060] Problems of inadequate or inappropriate gonadotropin levels havebeen recognized as causes of ovulatory dysfunction since the 1960s. Theclinical effectiveness of the various gonadotropin preparations used wasproportional to the amount of FSH administered. Initial evidencesuggested that exogenous LH is not required for adequatefolliculogenesis during ovulation induction. It became apparent,however, that women given only exogenous FSH fail to produce adequatefollicular estradiol for ovulation induction. The presence of at leastsome amount of exogenous or endogenous LH for ovulation induction in thehuman appears to be important. FIG. 1 illustrates a schematicrepresentation of a generalized ovulation induction regimen. An FSHpreparation is given at 75 IU/day for the first 7 days. At the end of 7days, an ultrasound scan is taken to assess follicular diameter andserum estradiol is measured. If the follicle is less than 12 mm, thedose of FSH is doubled, and a subsequent scan is taken in another 5-7days. Patients with follicles ≧15 mm diameter receive an ovulatory bolusdose of hCG.

III. Generalized Ovulation Induction Regimen

[0061] Ovulation induction is as much an art as a science. Despite aremarkable array of treatment protocols, no single approach or specificmethod is uniquely correct. Certain principles do apply, however, andprovide the basis for safe and effective treatment. However, it shouldbe noted that the criteria set forth below for inducing ovulation isgiven for example purposes only and may vary significantly by clinic,patient and the goal of the treatment.

[0062] The first cycle usually involves the administration of FSH dailybeginning on day 4 to 7 of a withdrawal bleed. Follicle growth andresponse are monitored by both estrogen levels and ultrasound. Adequatefollicle stimulation is usually achieved by 7 to 14 days of continuousFSH administration. Treatment with FSH for less than 8 days isassociated with increased spontaneous abortion rates among pregnantpatients.

[0063] Once sufficient follicle development has been achieved (two 16-to 18-mm follicles together with a progressive rise in serum estrogen to500 to 1,000 pg/mL), hCG (5,000 or 10,000 IU) is administered. Thetiming of hCG administration is important because the principalcomplications attributed to gonadotropin-induced hyperstimulation andmultiple pregnancies probably result from the prolonged effects of hCG.Although the half-life of hCG is approximately 8 hours, it may bedetectable in the patient's blood for 7 to 10 days after injection andmisdiagnosed as a successful treatment pregnancy. Followingadministration of hCG, the couple is instructed to have intercourse onthat night and once or twice more over the next 48 hours.

[0064] The fundamental rule regarding gonadotropin administration isthat each and every treatment cycle must be individualized, monitored,and adjusted appropriately. Monitoring is necessary not only to enhanceovulation and pregnancy rates but also to reduce the risk of severeovarian hyperstimulation and its potential consequences and multiplepregnancies.

[0065] This is achieved by frequent determinations of plasma/serumestradiol levels and by inspection of the ovaries by ultrasound.Estradiol levels generally correlate with the number of growingfollicles but not necessarily with the number of mature follicles.Reliance on estradiol levels as the marker for follicle maturity mayerroneously suggest follicle maturity in the presence of multiple smallfollicles, resulting in premature hCG administration. Because folliculargrowth correlates directly with ovum maturation, ultrasound assessmentof mean follicular diameter may be a better indicator in assessingmaturity and timing of hCG administration. Estrogen levels shouldtherefore be used to assess early follicular development as an indicatorof gonadotropin response, and ultrasound should be used to assess thenumber and size of maturing follicles.

[0066] The goal of most treatments is to maximize the potential for asingleton pregnancy while reducing the risk of hyperstimulationsyndrome. Estradiol levels between 1000 and 1500 pg/ mL appear to beoptimal, but actual levels may vary depending on the laboratory used andthe physician's experience. The risk of hyperstimulation increases withhigher estradiol levels. In general, when serum estradiol exceeds 2000pg/ mL, hCG should be administered with great caution or withheld toallow follicles to regress. In hypogonadotropic hypogonadism, the riskof severe hyperstimulation for values greater than 2400 pg/mL is 5 % inpregnancy cycles and 1 % in non-pregnant cycles. Furthermore, becausehyperstimulation tend to correlate with the number of follicles present,a decision to withhold hCG may be also based on an ultrasound finding of10 or more developing follicles.

[0067] The following treatises on female infertility, stimulatedfolliculogenesis and ovulation induction are incorporated by referenceherein: Reproductive Endocrinology, Surgery, and Technology, Volumes 1and 2; Editors: E. Y. Adashi et al (1996) and Shoham et al (1999).

IV. Methods of the Present Invention

[0068] The present invention relates to a method of ovulation inductionthat comprises the administration of a non-polypeptide cAMP levelmodulator in an ovulation induction regimen in which the modulator isadministered to enhance or substitute the administration of hCG (or LH),which is typically administered prior to the luteal phase of an inducedor naturally occurring ovulatory cycle. The modulator can beadministered alone or co-administered, either simultaneously orsequentially, with hCG (or LH), as well as by different modes ofdelivery (e.g., parenterally or orally).

[0069] In another aspect, the invention provides for theco-administration, either simultaneously or sequentially, of anon-polypeptide cAMP level modulator with LH or CG prior to the lutealphase of the female host's ovulatory cycle. In addition, since it isbelieved that the principal complications attributed to gonadotropininduced ovarian hyperstimulation and consequent multiple pregnanciesprobably result from the prolonged effects of hCG, the inventionprovides for the use of lower concentrations of LH or CG administered tothe host than concentrations that are used in existing ovulationinduction regimens and thereby lowering the likelihood of ovarianhyperstimulation, and consequently averting the adverse effectsassociated with that condition: multiple births, low weight newborns andhealth complications for the mother.

[0070] It should be noted that the administration of non-polypeptidecAMP level modulators have no therapeutic effect on follicularmaturation and development during the ovulatory cycle.

[0071] Thus, the present invention relates to methods of inducingovulation in a female host comprising the administration of anon-polypeptide cyclic adenosine monophosphate (cAMP) level modulator tothe female host. The non-polypeptide cAMP level modulators act, directlyor indirectly, to increase intracellular levels of cAMP. Such compoundscan increase cAMP levels by stimulating cAMP synthesis or by inhibitingits degradation, or both. Examples of modulators which increase thesynthesis of cAMP include activators of adenyl cyclase such asforskolin. Examples of modulators that decrease cAMP degradation includeinhibitors of phosphodiesterases such theophylline. Preferrednon-polypeptide cAMP level modulators include phosphodiesteraseinhibitors, particularly inhibitors of phosphodiesterase 4 isoforms.

[0072] In still another aspect, the invention provides for specificadministration of the non-polypeptide cAMP level modulator prior to theluteal phase of the host's ovulatory cycle. As an enhancer or substituteof hCG in an ovulation induction regimen, in which hCG is typicallyadministered towards the end of the follicular phase but prior to theluteal phase of the ovulatory cycle.

[0073] The preferred timing of administration of the non-polypeptidecAMP level modulator is prior to the luteal phase of the host'sovulatory cycle.

[0074] The invention also provides for the stimulation of folliculardevelopment prior to the administration of a non-polypeptide cAMP levelmodulator for inducing ovulation which comprises the administration ofan agent which increases follicular stimulating hormone (FSH)concentrations during the follicular phase of the host's ovulatorycycle. Preferred agents include FSH, itself, clomiphene, selectiveestrogen receptor modulators, aromatase inhibitors and agents which arenot FSH but have FSH biological function and activity. Thus,administration of the agents described herein in a prescribed timingrelative to the growth and maturation of the follicle are claimed toimprove the process of ovulation and subsequent fertilization that musttake place if conception is to occur.

[0075] It should be noted that when a non-polypeptide cAMP levelmodulator is administered alone and not co-administered with hCG, thepresent invention provides for the opportunity of earlier diagnostictesting for pregnancy than current ovulation induction regimensinvolving the use of CG.

V. Phosphodiesterase Inhibitors

[0076] For phosphodiesterase inhibitors used as non-polypeptide cAMPmodulators, essentially any non-toxic inhibitor of PDE can be used inthe methods of the invention, including selective and non-selectiveinhibitors of PDE4. Suitable non-selective inhibitors of PDE4 andcombined PDE3/PDE4 inhibitors include theophylline,isobutylmethylxanthine, AH-21-132, Org-30029 (Organon), Org-20241(Organon), Org-9731 (Organon), Zardaverine, vinpocetine, EHNA (MEP-1),Milrinone, Siguazodan, Zaprinast, SK+F 96231, Tolafentrine (Byk Gulden),Filaminast (Wyeth-Ayerst Pharmaceuticals).

[0077] Particularly preferred are selective inhibitors specific forPDE4. Many known selective PDE4 inhibitors fall into one of six chemicalstructural classes, rolipram-like, xanthines, nitraquazones,benzofurans, naphthalenes and quinolines. Examples of rolipram-likeanalogs include imidazolidinones and pyrrolizidinone mimetics ofrolipram and Ro 20-1724, as well as benzamide derivatives of rolipramsuch as RP 73401 (Rhone-Poulenc Rorer). Xanthine analogs includeDenbufylline (SmithKline Beecham)and Arofylline (Almirall); Nitraquazoneanalogs include CP-77,059 (Pfizer) and a series ofpyrid[2,3d]pyridazin-5-ones (Syntex); Benzofuran analogs includeEP-685479 (Bayer); Napthalene analogs include T-440 (Tanabe Seiyaku);and Quinoline analogs include SDZ-ISQ-844 (Novartis).

[0078] More preferred are the following:

[0079] Compounds disclosed in WO 97/42174 (Pfizer, Inc.):

[0080] wherein R¹ is cyclopentyl or cyclohexy, R² is ethyl, R³ is acarboxylic acid, ester or primary amide, hydroxymethyl or a carbonylgroup, and R⁴ is cyano;

[0081] Compounds disclosed in U.S. Pat. Nos. 5,710,160 and 5,710,170(Merck Frost Canada, Inc.):

[0082] wherein R is selected from H, CO-(4-mepiperazinyl),CO-(pyrrolidinyl), CHNOH, 5-tetrazolyl, (CH)OHCH₃, COCH₃, CONHSO₂CH₃,CONHSO₂Ph, CONHSO₂CF₃, CONHSO₂C₆H₄CH₃(o), CH₂CONHSO₂Ph, CH₂CONHSO₂CF₃,COH(CF₃)₂ and SO₂NHCOC₆H₄CH₃(o).

[0083] Compounds disclosed in WO 98/20007 (Darwin Discovery),represented by:

[0084] Compounds disclosed in WO 98/14432 (Janssen Pharm NV):

[0085] wherein R¹ is H or CH₃, R² is CH₃ or CHF₂, R³ is cyclopentyl,indanyl, cyclopropylmethyl, Ph(CH₂)₅, THF.

[0086] Compounds disclosed in WO 98/18796 (Novartis):

[0087] wherein X is NH₂, OH, NHPh, NPh₂, Ph, C₆H₄CO₂H and Y is Cl, CN,NO₂.

[0088] Compounds disclosed in WO 97/49702 (Pfizer, Inc.), representedby:

[0089] Compounds disclosed in WO 97/48697 (Rhône-Poulenc Rorer):

[0090] wherein R¹ is cyclopentyl, cyclopropyl, Ph(CH₂)₃, Benzyl or H, R²is CH₃, Benzyl, Tosyl or H, R³ is CH₃ or Cl, A is CH, C-alkyl, O or N,and B is C or N.

[0091] Compounds disclosed in WO 98/02440 (Bayer AG):

[0092] wherein R¹ is CH₃, OH, Oallyl, OCH₃,C₂H₅, Propyl or Acetyl, R² is3-Cl, 2,4-Cl₂, 3-NO₂, 3-Br, 4-F, 4-Cl, 2,4-(CH₃)₂ or 2,4 (CH₃O)₂.

[0093] Compounds disclosed in WO 97/44337, WO 97/44036, WO 97/44322(Chiroscience Ltd.):

[0094] wherein R¹ is CH₃, Ethyl or Propyl, R² and R³ are independentlyselected from H, optionally substituted aryl or heteroaryl, and R⁴ andR⁵ are independently selected from acyl, alkyl (optionally substitutedwith OH! or halogen), aryl, heteroaryl, and acyl substituted with arylor heteroaryl.

[0095] Compounds disclosed in U.S. Pat. No. 6,303,789 (Byk GuldenLomberg Chemische Fabrik GmbH):

[0096] wherein R¹ is 1-6C-alkoxy, 3-7C-cycloalkoxy,3-7C-cycloalkylmethoxy, benzyloxy or 1-4C-alkoxy which is completely orpredominantly substituted by fluorine; R² is hydrogen; and R ishydrogen; or R and R³ are together a methylene group; R⁴ is hydrogen,1-8C-alkyl, 1-6C-alkoxy-1-4C-alkyl, 1-6C-alkylthio-1-4-alkyl,1-6C-alkylsulfinyl-1-4C-alkyl, 1-6C-alkylsulfonyl-1-4C-alkyl,1-8C-alkylcarbonyl, 3-7C-cycloalkyl, 3-7C-cycloalkymethyl,phenyl-1-4C-alkyl or 1-4C-alkyl which is completely or predominantlysubstituted by fluorine; R⁵ is phenyl, pyridyl, phenyl substituted byR⁵¹, R⁵² and R⁵³ or pyridyl substituted by R⁵⁴, R⁵⁵, R⁵⁶ and R⁵⁷,wherein R⁵¹ is hydroxyl, halogen, cyano, carboxyl, trifluoromethyl,1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyl,1-4C-alkylcarbonyloxy, amino, mono- or di-1-4C-alkylamino or1-4C-alkylcarbonylamino; R⁵² is hydrogen, hydroxyl, halogen, amino,trifluoromethyl, 1-4C-alkyl or 1-4C-alkoxy, R⁵³ is hydrogen, halogen,1-4C-alkyl or 1-4C-alkoxy; R⁵⁴ hydroxyl, halogen, cyano, carboxyl,1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl or amino; R⁵⁵ is hydrogen,halogen, amino or 1-4C-alkyl; R⁵⁶ is hydrogen or halogen; and R⁵⁷ ishydrogen or halogen; n is 1 or 2; m is 1 or 2; where the sum of m and nmay only assume the values 2 or 3, a salt of these compounds and theN-oxide of the pyridines or a salt thereof.

[0097] Compounds disclosed in U.S. Pat. No. 6,316,472 (Merck FrosstCanada):

[0098] or a pharmaceutically acceptable salt or hydrate thereof wherein:

[0099] Y represents N or N-oxide;

[0100] y represents 0, 1 or 2;

[0101] R¹ and R² are independently selected from H, C₁₋₆ alkyl andhaloC₁₋₆ alkyl;

[0102] R³ and R⁴ are independently selected from H and C.sub.1-6 alkyl,or R.sup.3 and R⁴ attached to the same carbon atom taken togetherrepresent a carbonyl oxygen atom, or

[0103] R³ and R⁴ attached to different carbon atoms considered incombination with the carbon atoms to which they are attached along withany intervening atoms and represent a saturated 5, 6- or 7-memberedcarbocyclic ring;

[0104] R⁵ and R.sup.6 independently represent a member selected from thegroup consisting of: H, C.sub.1-6 alkyl, haloC.sub.1-6 alkyl and CN;

[0105] n represents an integer of from 0-6;

[0106] Ar¹ is an aromatic group.

[0107] Particularly preferred are the following: Rolipram, Arofylline(Almirall), Ariflo® (SmithKline Beecham), Roflumilast (Byk Gulden),Denbufylline (SmithKline Beecham), RS-17597 (Syntex), SDZ-ISQ-844(Novartis),4-[2,3-bis(hydroxymethyl)-6,7-diethoxynaphthyl]-1-(2-hydroxyethyl)hydropyridin-2-one(T-440; Tanabe Seiyaku), methyl3-[6-(2H-3,4,5,6-tetrahydropyran-2-yloxy)-2-(3-thienylcarbonyl)benzo[b]furan-3-yl]propanoate(Bayer),2-methyl-1-[2-(methylethyl)(8-hydropyrazolo[1,5-a]pyridin-3-yl)]propan-1-one(Ibudilast; Kyorin),N-(3,5-dichloro(4-pyridyl))(3-cyclopentyloxy-4-methoxyphenyl)carboxamide(RP 73401; Rhône-Poulenc Rorer),(1E)-1-aza-2-(3-cyclopentyloxy-4-methoxyphenyl)prop-1-enyl aminooate(PDA-641; American Home Products),4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexanecarboxylic acid(SB207499; SmithKline Beecham), Cipamfylline (SmithKline Beecham),5-[3-((2S)bicyclo[2.2.l]hept-2-yloxy)-4-methoxyphenyl]-1,3-diazaperhydroin-2-one(CP-80633; Pfizer), 1-(3-nitrophenyl)-3-5(3-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidine-2,4-dione(RS-25344; Syntex),4-((1R)-1-phenyl-2-(3-pyridyl)ethyl)-2-cyclopentyloxy-1-methoxybenzene(CDP-840; Celltech),(3-{[(3-cyclopentyloxy-4-methoxyphenyl)methyl]amino}pyrazol-4-yl)methan-1-ol,Ro-20-1724 (Roche Holding AG), Piclamilast, Doxofylline (InstitutoBiologico Chemioterapico ABC SpA), RPR-132294 (Rhône-Poulenc Rorer),RPR-117658A (Rhône-Poulenc Rorer), L-787258 (Merck Frosst Canada),E-4021 (Eisai Co. Ltd.), GF-248 (Glaxo Wellcome), SKF-107806 (SmithKlineBeecham), IPL-4088 (Inflazyme Pharmaceuticals Ltd.),{3-[(3-cyclopentyloxy-4-methoxyphenyl)methyl]-8-(methylethyl)purin-6-yl}ethylamine(V-11294A; Napp Research Centre Ltd.), Atizoram (Pfizer),5-(3-cyclopentyloxy-4-methoxyphenyl)pyridine-2-carboxamide (CP-353164;Pfizer), methyl3-[2,4-dioxo-3-benzyl-1,3-dihydropyridino[2,3-d]pyrimidinyl]benzoate(CP-77059; Pfizer), CP-146523 (Pfizer), CP-293321 (Pfizer),CI-1044(Pfizer), PD-189659(Pfizer), CI-1018 (Pfizer), CP-220629(Pfizer),1-(3-nitrophenyl)-3-(4-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidine-2,4-dione(RS-25344-000; Roche Bioscience), Mesopram (Schering AG),N-(2,5-dichloro(3-pyridyl))(8-methoxy(5-quinolyl))carboxamide (D-4418;Chiroscience), T-2585 (Tanabe Seiyaku),4-[4-methoxy-3-(5-phenylpentyloxy)phenyl]-2-methylbenzoic acid, XT-044(Hokuriku University), XT-611 (Kanzawa University), WAY-126120(Wyeth-Ayerst Pharmaceuticals Inc.),1-aza-10-(3-cyclopentyloxy-4-methoxyphenyl)-7,8-dimethyl-3-oxaspiro[4.5]dec-7-en-2-one(WAY-122331; Wyeth-Ayerst Pharmaceuticals Inc.),[(3S)-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl-5-oxopyrazolidinyl]-N-(3-pyridylmethyl)carboxamide(WAY-127093B; Wyeth-Ayerst Pharmaceuticals Inc.), PDB-093 (Wyeth-AyerstPharmaceuticals Inc.),3-(1,3-dioxobenzo[c]azolin-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)propanamide(CDC-801; Celgene Corp.), CC-7085 (Celgene Corp.), CDC-998 (CelgeneCorp.), NCS-613 (CNRS), CH-3697 (Chiroscience), CH-3442 (Chiroscience),CH-2874 (Chiroscience), CH-4139 (Chiroscience), RPR-114597(Rhône-Poulenc Rorer), RPR-122818 (Rhône-Poulenc Rorer), (7aS,7R)-7-(3-cyclopentyloxy-4-methoxyphenyl)-7a-methyl-2,5,6,7,7a-pentahydro-2-azapyrrolizin-3-one,GW-3600 (Glaxo-Wellcome), KF-19514 (Kyowa Hakko Kogyo Co Ltd.), CH-422(Celltech Group), CH-673 (Celltech Group), CH-928 (Celltech Group),D-22888 (Asta Medica), AWD-12-232 (Asta Medica), YM-58997 (Yamanouchi),IC-485 (ICOS Corp.), KW-4490 (Kyowa Hakko Kogyo Co. Ltd.), YM-976(Yamanouchi), Sch-351591 (Celltech Group), AWD-12-343 (Asta Medica),N-(3,5-dichloro(4-pyridyl))-2-{1-[(4-fluorophenyl)methyl]-5-hydroxyindolin-3-yl}-2-oxoacetamide(AWD-12-281; Asta Medica), Ibudilast (Kyorin Pharmaceutical Co. Ltd.),Cilomilast (SmithKline Beecham), BAY-19-8004 (Bayer), methyl3-{2-[(4-chlorophenyl)carbonyl]-6-hydroxybenzo[b]furan-3-yl}propanoate,5-methyl-4-[(4-methylthiophenyl)carbonyl]-4-imidazolin-2-one,5,6-diethoxybenzo[b]thiophene-2-carboxylic acid (Tibenelast), and4-(3-bromophenyl)-1-ethyl-7-methylhydropyridino[2,3-b]pyridin-2-one(YM-58897; Yamanouchi).

[0108] In one embodiment, the invention provides for the use of a PDEinhibitor, preferably a PDE4 inhibitor, for triggering ovulation afterfollicular growth and maturation has been induced with FSH. Also withinthe scope of the invention is the use of a PDE inhibitor, particularly aPDE4 inhibitor, for triggering ovulation after follicular growth andmaturation has been induced with a compound or preparation having FSHactivity. A particularly preferred substitute for or adjuvant to FSHtreatment is an aromatase inhibitor, for example, YM-511 (Yamanouchi),Letrozole (Novartis), Anastrozole (AstraZeneca) or Fadrozole (Novartis).In a preferred regimen for assisted reproductive technologies (ART), inwhich it is desired to obtain multiple oocytes for in vitrofertilization, patients are administered an aromatase inhibitor (e.g.,at or about 2.5-5 mg/day of Letrozole, or Anastrozole) from at or aboutday 3 to at or about day 7, or from at or about day 3 to at or about day8 of the menstrual cycle, together with at or about 50-225, preferable50-150 IU FSH/ day, starting on or about day 3 to day 7 of the menstrualcycle, FSH continuing until there are at least two leading follicleshaving a mean diameter of greater than or equal to at or about 16 mm. Atthis point, a PDE inhibitor, preferably a PDE4 inhibitor is administeredin a dose sufficient to trigger ovulation.

[0109] Alternatively, the aromatase inhibitor may be used as the solefollicle growth stimulating agent (i.e., in the absence of FSH), byusing a higher dose of aromatase inhibitor (e.g., 2-10 mg/day ofLetrozole or Anastrozole) and/or by prolonging administration (e.g.,days3 to 8, 3 to 9, or 3 to 10). When follicular maturation is judgedsufficient by sonography, an ovulation triggering dose of PDE inhibitor,preferably PDE4 inhibitor is given. This regimen permits the collectionof multiple oocytes, while avoiding injections, as all the agents usedare orally available.

[0110] In ovulation induction, it is desirable to cause the release ofonly one ovum. This can be achieved, according to the invention, usingFSH to stimulate follicular growth and maturation, followed byadministration of a PDE inhibitor, preferably a PDE4 inhibitor, totrigger ovulation. Also within the scope of the invention are ovulationinduction regimens in which follicular growth and maturation is inducedwith a substitute for FSH, for example an aromatase inhibitor.

[0111] In a preferred regimen for ovulation induction, a patient isadministered a dose of aromatase inhibitor (e.g., 2.5-5 mg/day ofLetrozole or Anastrozole) from at or about day 3 to at or about day 7,or from at or about day 5 to at or about day 9 of the menstrual cycle(in the absence of FSH). Alternatively, a single dose of aromataseinhibitor may be given (e.g., 5-30 mg of Letrozole or Anastrozole,preferably 10 or 20 mg), at or about day 3 or day 4 of the menstrualcycle. Ovulation is triggered with an ovulation triggering dose of a PDEinhibitor preferably a PDE4 inhibitor. This regimen provides anovulation induction protocol that requires no injections.

VI. Pharmaceutical Compositions

[0112] The non-polypeptide cAMP level modulators and agents whichincrease FSH concentrations in a female host (also referred to herein as“active compounds”) of the invention, and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the active compounds and a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0113] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, (e.g., intravenous, intradermal,subcutaneous), oral, inhalation, transdermal (topical), transmucosal,and rectal administration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0114] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0115] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0116] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0117] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0118] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0119] The preferred route of administration for non-polypeptide cAMPlevel modulators including phosphodiesterase inhibitors, as well as forclomiphene, selective estrogen receptor modulators, aromatase inhibitorsand inhibitors of steroidogenic enzymes is by oral administration. Theseactive compounds also can be administered subcutaneously by injection,intravenously or trans-vaginally (for local administration). Thepreferred route of administration of FSH, LH or hCG is by subcutaneousinjection, but could also be administered intravenously.

[0120] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal or vaginal delivery.

[0121] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

[0122] Compounds of the invention can be employed, either alone or incombination with one or more other therapeutic agents as discussedabove, as a pharmaceutical composition in mixture with conventionalexcipient, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for oral, parenteral, enteral or topicalapplication which do not deleteriously react with the active compoundsand are not deleterious to the recipient thereof. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

[0123] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0124] It will be appreciated that the actual preferred amounts ofactive compounds used in a given therapy will vary according to thespecific compound being utilized, the particular compositionsformulated, the mode of application, the particular site ofadministration, etc. Optimal administration rates for a given protocolof administration can be readily ascertained by those skilled in the artusing conventional dosage determination tests conducted with regard tothe foregoing guidelines. See also Remington's Pharmaceutical Sciences.In general, a suitable effective dose of one or more compounds of theinvention, particularly when using the more potent compound(s) of theinvention, will be in the range of from 0.01 to 100 milligrams perkilogram of bodyweight of recipient per day, preferably in the range offrom 0.01 to 20 milligrams per kilogram bodyweight of recipient per day,more preferably in the range of 0.05 to 4 milligrams per kilogrambodyweight of recipient per day. The desired dose is suitablyadministered once daily, or several sub-doses, e.g., 2 to 4 sub-doses,are administered at appropriate intervals through the day, or otherappropriate schedule. Such sub-doses may be administered as unit dosageforms, e.g., containing from 0.05 to 10 milligrams of compound(s) of theinvention, per unit dosage.

[0125] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0126] The following non-limiting examples are illustrative of theinvention.

VII. Exemplification

[0127] An in vivo ovulation model was been developed in which FSH isadministered to immature rats bid x 2 or 3 days to induce follicularmaturation, followed by a single ovulatory dose of hCG. A singleinjection of non-polypeptide CAMP level modulators (e.g., Compound 1,Compound 2, etc.) co-administered with a sub-ovulatory dose of hCG orinjected alone resulted in an induction of ovulation. These results areconsistent with a model in which increased cAMP levels enhance orsubstitute for hCG but not FSH. The role of FSH in any ovulationinduction regimen is for promoting follicular development andmaturation, not ovulation induction.

[0128] The Compounds of Examples 1 through 9 are identified as follows:Compound 1 is Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylic acid; Compound 2 is3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide;Compound 3 is 2-(4-(6,7-Diethoxy-2,3-bis (hydroxymethyl)naphthalen-1-yl) pyridin-2-yl)-4-(3-pyridyl) pthalazin-1 (2H)-onehydrochloride; and Compound 4 is7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.

Example 1: Effect of Compounds 1 and Compounds 2 on Rat OvarianGranulosa Cell cAMP Levels, Alone or with Gonadotropins In Vitro

[0129] Ovaries were removed from immature 25 day old, hypophysectomized,diethylstilbesterol treated Sprague-Dawley rats. The ovaries wererepeatedly punctured with 27 gauge needles to liberate granulosa cellsfrom the follicles. Cells were washed and re-suspended in McCoys 5Amedia+0.1% BSA+2 μM androstenedione. Viable cells in number of 100,000were loaded into 6-well tissue culture dishes in a 1.0 ml volume (withCompound 1 and Compound 2 at a concentration of 25 micromolar, eitheralone or in conjunction with a low, 0.1 pM dose of gonadotropin). Plateswere incubated in a 37° C. incubator, 100% humidity, 5.0% CO₂ for 48hrs. Conditioned media were assayed in a cAMP specific RIA. Results areexpressed as mean plus or minus standard deviations. As seen in FIG. 2,Compounds 1 and 2 cause a significant increase in cAMP levels in thepresence of sub-effective concentrations of gonadotropin.

Example 2: Effect of the PDE Inhibitor Compound 1 on Follicle MaturationIn Vivo

[0130] Mature ovarian follicles generated in immature female rats bytreatment with a sub-optimal dose of FSH (1.08 IU/rat/injection; bid x 3days) with and without co-administration of Compound 1 (0.1, 1, 10, and50 mg/kg/injection: bid X 3 days). A single injection of an ovulatorydose of hCG (20 IU) was administered with the final FSH injection. ThePDE inhibitors were given in conjunction with sub-optimal doses of FSH.

[0131] All injections were subcutaneously administered. Ovulation wasdetermined 18 hours after hCG administration by counting oocytes inoviduct. Results are expressed as mean plus or minus standarddeviations. As seen in FIG. 3, data represent average number of oocytesin oviducts of all rats in each group and frequency of ovulating rats.As also noted in FIG. 3, a PDE inhibitor (Compound 1) inhibited (ratherthan stimulated) both ovulation administered at 50 mg/kg. The resultsdemonstrate that increasing doses of PDE inhibitor failed to enhance theability of a sub-optimal dose of FSH to prepare follicles to ovulate.

Example 3: Effect of the PDE Inhibitor Compound 1 on Ovulation, in thePresence of a Sub-Effective Dose of hCG In Vivo

[0132] Mature ovarian follicles generated in immature female rats bytreatment with an effective dose of FSH (2.16 IU/rat/injection; bid x 2days) were induced to ovulate with a single injection of hCG. hCG wasadministered at a sub-effective dose (3 IU) with and without a singleinjection of Compound 1 (50, 10 and 1 mg/kg) at the time of the finalFSH injection. Ovulation was determined 18 hours after hCGadministration by counting the number of ova in the oviduct. As seen inFIG. 4, a single injection of Compound 1 co-administered with asub-ovulatory dose of hCG resulted in an induction of ovulation. Allinjections were subcutaneous. Results are expressed as mean plus orminus standard deviations. This data demonstrates that a non-polypeptidecAMP level modulator, in this case a PDE inhibitor enhanceshCG-stimulated ovulation when a sub-optimal dose of hCG is administered.The effects of Compound 1, a known PDE inhibitor, are shown.

Example 4: Effect of PDE Inhibitor Compound 1 on Ovulation in thePresence or Absence of a Sub-Effective Dose of hCG In Vivo

[0133] Following FSH induced follicular maturation (2.16IU/rat/injection; bid X 2 days) Compound 1 was injected with and withouta sub-effective dose of hCG. Ovulation was determined 18 hours afterhCG/Compound 1 administration by counting oocytes in oviduct. Datarepresent average number of oocytes in oviducts of all rats in eachgroup and frequency of ovulating rats. As seen in FIG. 5, a singleinjection of Compound 1 administered alone, without a sub-ovulatory doseof hCG, resulted in an induction of ovulation in FSH pretreated rats.Results are expressed as mean plus or minus standard deviations. Thisdata demonstrates that a non-polypeptide cAMP level modulator, in thiscase a PDE inhibitor, Compound 1, is able to induce ovulation in theabsence of any injected hCG. Previous experiments, and those presentedhere have shown that follicles prepared with these doses of FSH do notovulate spontaneously, but require subsequent hCG administration.

Example 5: Effect of PDE Inhibitor Compound 2 on Ovulation in thePresence or Absence of a Sub-Effective Dose of hCG In Vivo

[0134] Following FSH induced follicular maturation (2.16IU/rat/injection; bid X 2 days) Compound 2 was injected with and withouta sub- effective dose of hCG. Ovulation was determined 18 hours afterhCG/Compound 2 administration by counting oocytes in oviduct. Datarepresent average number of oocytes in oviducts of all rats in eachgroup and frequency of ovulating rats. Results are expressed as meanplus or minus standard deviations. As seen in FIG. 6, a single injectionof Compound 2 administered alone, without a sub-ovulatory dose of hCG,resulted in an induction of ovulation in FSH pretreated rats. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 2, is able to induce ovulation in the absence ofany injected hCG.

Example 6: Effect of PDE Inhibitor Compound 2 on Ovulation In VivoFollowing Oral and Subcutaneous Administration

[0135] Following FSH induced follicular maturation (2.16IU/rat/injection; bid X 2 days) Compound 2 was either injectedsubcutaneously (subcutis) or administered by oral gavage. Ovulation wasdetermined 18 hours after Compound 2 administration by counting oocytesin oviduct. Data represent average number of oocytes in oviducts of allrats in each group and frequency of ovulating rats. Results areexpressed as mean plus or minus standard deviations. A s seen in FIG. 7,administration of Compound 2 by either subcutaneous or oral routeresulted in an induction of ovulation in FSH pretreated rats. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 2, is able to induce ovulation when administeredorally.

Example 7: Effect of PDE Inhibitor Compound 3 on Ovulation In VivoFollowing Oral and Subcutaneous Administration

[0136] Following FSH induced follicular maturation (2.16IU/rat/injection; bid X 2 days) Compound 3 was either injectedsubcutaneously (subcutis) or administered by oral gavage. Ovulation wasdetermined 18 hours after Compound 2 administration by counting oocytesin oviduct. Data represent average number of oocytes in oviducts of allrats in each group and frequency of ovulating rats. Results areexpressed as mean plus or minus standard deviations. As seen in FIG. 8,administration of Compound 3 by either subcutaneous or oral routeresulted in an induction of ovulation in FSH pretreated rats. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 3, is able to induce ovulation when administeredorally.

Example 8: Effect of PDE Inhibitor Compound 4 on Ovulation in thePresence or Absence of a Sub-Effective Dose of hCG In Vivo

[0137] Following FSH induced follicular maturation (2.16IU/rat/injection; bid X 2 days) Compound 4 was injected subcutaneouslywith and without a sub-effective dose of hCG. Ovulation was determined18 hours after hCG/Compound 4 administration by counting oocytes inoviduct. Data represent average number of oocytes in oviducts of allrats in each group and frequency of ovulating rats. As seen in FIG. 9, asingle injection of Compound 4 administered with a sub-ovulatory dose ofhCG resulted in an induction of ovulation in FSH pretreated rats.Compound 4 administered alone induced little or no ovulation. Resultsare expressed as mean plus or minus standard deviations. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 4, which is insufficient to induce ovulationalone, is able to induce ovulation in the presence of a sub-effectivedose of hCG.

Example 9: Effect of PDE Inhibitor Compound 2 on Ovulation and Fertility

[0138] Follicular maturation was induced in immature rats with FSH (4.33IU/rat/injection; bid X 2 days) and Pregnant Mare's Serum Gonadotropin(PMSG) (1.5 IU/rat/injection; bid X 2 days). This combination waspreviously found to induce follicular maturation and promote matingbehavior while maintaining a low spontaneous ovulation rate in theabsence of an additional injection of hCG. FSH and PMSG induced ratswere then treated with either Compound 2 (12 rats) or vehicle (12 rats)by subcutaneous administration. In one cohort of rats (6 rats pertreatment), ovulation was determined 18 hours after Compound 2 orvehicle administration by counting oocytes in oviduct. To assessfertility, another cohort of rats (6 rats per treatment) wereindividually placed overnight in a cage together with a single adultmale rat of proven fertility. The next day, male rats were removed andthe females were caged together by group until the day of parturition.The number of live full-term pups observed at the time of parturitionwas recorded. As seen in FIG. 10, a single injection of Compound 2resulted in an induction of ovulation in FSH/PMSG pretreated rats. Inaddition, rats treated with Compound #2 had an increased number of livepups at the time of parturition. Results for both ovulation and livepups are expressed as mean plus or minus standard deviations. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 2, induces ovulation of oocytes which arecapable of being fertilized in vivo.

[0139] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. The entire contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

[0140] Adashi et al (Eds.) Reproductive Endocrinology, Surgery, andTechnology, Volumes 1 and 2, Lippincott-Raven Publishers, Philadelphia(1996)

[0141] Bär T, “Benzamides with Tetrahydrofuranyloxy Substituents asPhosphodiesterase 4 Inhibitors”, U.S. Pat. No. 6,303,789, issued Oct.16, 2001

[0142] Bräunlich et al, “3-Ureido-Pyridofurans and Pyridothiophenes forthe Treatment of Inflammatory Processes”, international publication WO98/02440 of Jan. 22, 1998

[0143] Chappel S, “Novel Method of Ovulation Induction in Humans”, U.S.Pat. No. 5,087,615, issued Feb. 11, 1992

[0144] Cooke G M, “Differential effects of trilostane and cyanoketone onthe 3 beta-hydroxysteroid dehydrogenase-isomerase reactions in androgenand 16-androstene biosynthetic pathways in the pig testis”, J. SteroidBiochem Mol Biol 58:1 95-101 (1996)

[0145] Frenette et al, “Heterosubstituted Pyridine Derivatives as PDE 4Inhibitors”, U.S. Pat. No. 6,316,472, issued Nov. 13, 2001

[0146] Freyne et al, “PDE IV Inhibiting 2-CyanoiminoimidazoleDerivatives”, international publication WO 98/14432 of Apr. 9, 1998

[0147] Guay et al, “Diphenyl Pyridyl Ethane Derivatives as PDE IVInhibitors”, U.S. Pat. No. 5,710,160, issued Jan. 20, 1998

[0148] Guay et al, “Tri-aryl Ethane Derivatives as PDE IV Inhibitors”,U.S. Pat No. 5,710,170, issued Jan. 20, 1998

[0149] Hersperger R, “Naphthyridine Derivatives”, internationalpublication WO 98/18796 of May 6, 1998

[0150] Cox et al, “Substituted Azabicyclic Compounds and Their Use asInhibitors of the Production of TNF and Cyclic AMP Phosphodiesterase”,international publication WO 97/48697 of Dec. 24, 1997

[0151] Dyke et al, “Quinoilne Carboxamides as TNF Inhibitors and asPDE-IV Inhibitors”, international publication WO 97/44036 of Nov. 27,1997

[0152] Dyke et al, “Quinoline Sulfonamides as TNF Inhibitors and asPDE-IV Inhibitors”, international publication WO 97/44322 of Nov. 27,1997

[0153] Dyke et al, “Benzofuran Carboxamides and Their Therapeutic Use”,international publication WO 97/44337 of Nov. 27, 1997

[0154] Dyke et al, “Quinolines and Their Therapeutic Use”, internationalpublication WO 98/20007 of May 14, 1998

[0155] Marfat, A., “Substituted Indazole Derivatives and Their Use asInhibitors Phosphodiesterase (PDE) Type IV and the Production of TumorNecrosis Factor (TNF), international publication Wo 97/42174 of Nov. 13,1997

[0156] Marfat A, “Substituted Indazole Derivatives and Their Uses asPhosphodiesterase (PDE) Type IV and Tumor Necrosis Factor (TNF)Inhibitors”, international publication WO 97/49702 of Dec. 31, 1997

[0157] Shetty et al, “Effect of estrogen deprivation on the reproductivephysiology of male and female primates”, J. Steroid Biochem Mol Biol61:(3-6):157-166 (1997)

[0158] Shoham et al (Eds.) Female Infertility Therapy Current Practice;Martin Dunitz Ltd, London (1999)

[0159] Shughrue et al, “Regulation of progesterone receptor messengerribonucleic acid in the rat medial preoptic nucleus by estrogenic andantiestrogenic compounds: an in situ hybridization study”,Endocrinology, 1999 138(12):5476-5484 (1999)

[0160] Turner et al, “Effect of chronic administration of an aromataseinhibitor to adult male rats on pituitary and testicular function andfertility”, J. Endocrinol 164(2):225-238 (2000)

What is claimed is:
 1. A method of ovulation induction in a female hostcomprising the administration of a non-polypeptide cAMP level modulatorto said host.
 2. A method of claim 1 wherein said cAMP level modulatoris a phosphodiesterase inhibitor.
 3. A method of claim 2 wherein saidphosphodiesterase inhibitor is an inhibitor of a phosphodiesterase 4isoform.
 4. A method of ovulation induction in a female host comprisingthe administration of a non-polypeptide cAMP level modulator to saidhost prior to the luteal phase of the host's ovulatory cycle.
 5. Amethod of claim 4 wherein said non-polypeptide cAMP level modulator is aphosphodiesterase inhibitor.
 6. A method of claim 5 wherein saidphosphodiesterase inhibitor is an inhibitor of a phosphodiesterase 4isoform.
 7. A method of a combined treatment for stimulating folliculardevelopment and ovulation induction in a female host comprising theadministration of an agent which increases follicle stimulating hormoneconcentrations in said host during the follicular phase of the host'sovulatory cycle and administering a non-polypeptide cAMP level modulatorto said host prior to the luteal phase of the host's ovulatory cycle. 8.A method of claim 7 wherein said agent is follicle stimulating hormone.9. A method of claim 7 wherein said agent is clomiphene.
 10. A method ofclaim 7 wherein said agent is a selective estrogen receptor modulator.11. A method of claim 7 wherein said agent is an aromatase inhibitor.12. A method of claim 7 wherein said agent is an inhibitor of relatedsteroidogenic enzymes that results in a decrease in total estrogenproduction.
 13. A method of claim 7 wherein said non-polypeptide cAMPlevel modulator is a phosphodiesterase inhibitor.
 14. A method of claim13 wherein said phosphodiesterase inhibitor is an inhibitor of aphosphodiesterase 4 isoform.
 15. A method of claim 7 wherein luteinizinghormone is also administered to said host to induce ovulation prior tothe luteal phase of the host's ovulatory cycle.
 16. A method of claim 7wherein luteinizing hormone is also administered at reducedconcentrations compared to existing regimens to said host to induceovulation prior the luteal phase of the host's ovulatory cycle.
 17. Amethod of claim 7 wherein chorionic gonadotropin is also administered tosaid host to induce ovulation prior the luteal phase of the host'sovulatory cycle.
 18. A method of claim 7 wherein chorionic gonadotropinis also administered at reduced concentrations compared to existingregimens to said host to induce ovulation prior to the luteal phase ofthe host's ovulatory cycle.
 19. A method of ovulation induction in afemale host comprising the administration of a non-polypeptide cAMPlevel modulator to said host at the time point of an existing ovulationinduction regimen at which hCG or LH is administered to said host.
 20. Amethod of claim 20 wherein the non-polypeptide cAMP level modulator isco-administered with hCG or LH.
 21. A method of claim 20 wherein thenon-polypeptide cAMP level modulator is administered alone and notco-administered with hCG or LH.
 22. A non-polypeptide cAMP levelmodulator for its use as an ovulation induction agent.
 23. Anon-polypeptide cAMP level modulator for its use in the treatment of ananovulation disorder.
 24. A pharmaceutical composition containingnon-polypeptide cAMP level modulator, for its use in the treatment of ananovulation disorder.
 25. Use of non-polypeptide cAMP level modulator ina pharmaceutical composition for the treatment of an anovulatorydisorder.
 26. Use of non-polypeptide cAMP level modulator for thepreparation of a medicament to be used in the treatment of ananovulatory disorder.
 27. A method of collecting oocytes for in vitrofertilization comprising the administration of a non-polypeptide cAMPlevel modulator.
 28. A method of claim 2 wherein the phosphodiesteraseinhibitor is selected from the group consisting of: Rolipram, Arofylline(Almirall), Ariflo® (SmithKline Beecham), Roflumilast (Byk Gulden),Denbufylline (SmithKline Beecham), RS-17597 (Syntex), SDZ-ISQ-844(Novartis),4-[2,3-bis(hydroxymethyl)-6,7-diethoxynaphthyl]-1-(2-hydroxyethyl)hydropyridin-2-one(T-440; Tanabe Seiyaku), methyl3-[6-(2H-3,4,5,6-tetrahydropyran-2-yloxy)-2-(3-thienylcarbonyl)benzo[b]furan-3-yl]propanoate(Bayer),2-methyl-1-[2-(methylethyl)(8-hydropyrazolo[l,5-a]pyridin-3-yl)]propan-1-one(Ibudilast; Kyorin),N-(3,5-dichloro(4-pyridyl))(3-cyclopentyloxy-4-methoxyphenyl)carboxamide(RP 73401; Rhône-Poulenc Rorer),(1E)-1-aza-2-(3-cyclopentyloxy-4-methoxyphenyl)prop-1-enyl aminooate(PDA-641; American Home Products),4-cyano-4-(3-cyclopentyloxy-4-ethoxyphenyl)cyclohexanecarboxylic acid(SB207499; SmithKline Beecham), Cipamfylline (SmithKline Beecham),5-[3-((2S)bicyclo[2.2.1]hept-2-yloxy)-4-methoxyphenyl]-1,3-diazaperhydroin-2-one(CP-80633; Pfizer),1-(3-nitrophenyl)-3-(3-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidine-2,4-dione(RS-25344; Syntex), 4-((1R)-1-phenyl-2-(4-pyridyl)ethyl)-2-cyclopentyloxy-l-methoxybenzene (CDP-840; Celltech),(3-{[(3-cyclopentyloxy-4-methoxyphenyl)methyl]amino}pyrazol-4-yl)methan-1-ol,Ro-20-1724 (Roche Holding AG), Piclamilast, Doxofylline (InstitutoBiologico Chemioterapico ABC SpA), RPR-132294 (Rhône-Poulenc Rorer),RPR-117658A (Rhône-Poulenc Rorer), L-787258 (Merck Frosst Canada),E-4021 (Eisai Co. Ltd.), GF-248 (Glaxo Wellcome), SKF-107806 (SmithKlineBeecham), IPL-4088 (Inflazyme Pharmaceuticals Ltd.),{3-[(3-cyclopentyloxy-4-methoxyphenyl)methyl]-8-(methylethyl)purin-6-yl}ethylamine(V-11294A; Napp Research Centre Ltd.), Atizoram (Pfizer),5-(3-cyclopentyloxy-4-methoxyphenyl)pyridine-2-carboxamide (CP-353164;Pfizer), methyl3-[2,4-dioxo-3-benzyl-1,3-dihydropyridino[2,3-d]pyrimidinyl]benzoate(CP-77059; Pfizer), CP-146523 (Pfizer), CP-293321 (Pfizer),CI-1044(Pfizer), PD-189659(Pfizer), CI-1018 (Pfizer), CP-220629(Pfizer),1-(3-nitrophenyl)-3-(4-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidine-2,4-dione(RS-25344-000; Roche Bioscience), Mesopram (Schering AG),N-(2,5-dichloro(3-pyridyl))(8-methoxy(5-quinolyl))carboxamide (D-4418;Chiroscience), T-2585 (Tanabe Seiyaku),4-[4-methoxy-3-(5-phenylpentyloxy)phenyl]-2-methylbenzoic acid, XT-044(Hokuriku University), XT-611 (Kanzawa University), WAY-126120(Wyeth-Ayerst Pharmaceuticals Inc.),1-aza-10-(3-cyclopentyloxy-4-methoxyphenyl)-7.8-dimethyl-3-oxaspiro[4.5]dec-7-en-2-one(WAY-122331; Wyeth-Ayerst Pharmaceuticals Inc.),[(3S)-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl-5-oxopyrazolidinyl]-N-(3-pyridylmethyl)carboxamide(WAY-127093B; Wyeth-Ayerst Pharmaceuticals Inc.), PDB-093 (Wyeth-AyerstPharmaceuticals Inc.),3-(1,3-dioxobenzo[c]azolin-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)propanamide(CDC-801; Celgene Corp.), CC-7085 (Celgene Corp.), CDC-998 (CelgeneCorp.), NCS-613 (CNRS), CH-3697 (Chiroscience), CH-3442 (Chiroscience),CH-2874 (Chiroscience), CH-4139 (Chiroscience), RPR-114597(Rhône-Poulenc Rorer), RPR-122818 (Rhône-Poulenc Rorer), (7aS,7R)-7-(3-cyclopentyloxy-4-methoxyphenyl)-7a-methyl-2,5,6,7,7a-pentahydro-2-azapyrrolizin-3-one,GW-3600(Glaxo-Wellcome), KF-19514 (Kyowa Hakko Kogyo Co Ltd.), CH-422 (CelltechGroup), CH-673 (Celltech Group), CH-928 (Celltech Group), D-22888 (AstaMedica), AWD-12-232 (Asta Medica), YM-58997 (Yamanouchi), IC-485 (ICOSCorp.), KW-4490 (Kyowa Hakko Kogyo Co. Ltd.), YM-976 (Yamanouchi),Sch-351591 (Celltech Group), AWD-12-343 (Asta Medica),N-(3,5-dichloro(4-pyridyl))-2-{1-[(4-fluorophenyl)methyl]-5-hydroxyindolin-3-yl}-2-oxoacetamide(AWD-12-281; Asta Medica), Ibudilast (Kyorin Pharmaceutical Co. Ltd.),Cilomilast (SmithKline Beecham), BAY-19-8004 (Bayer), methyl3-{2-[(4-chlorophenyl)carbonyl]-6-hydroxybenzo[b]furan-3-yl}propanoate,5-methyl-4-[(4-methylthiophenyl)carbonyl]-4-imidazolin-2-one,5,6-diethoxybenzo[b]thiophene-2-carboxylic acid (Tibenelast), and4-(3-bromophenyl)-1-ethyl-7-methylhydropyridino[2, 3-b]pyridin-2-one(YM-58897; Yamanouchi).
 29. A method of claim 2 wherein thephosphodiesterase inhibitor is selected from the group consisting of:theophylline, isobutylmethylxanthine, AH-21-132, Org-30029 (Organon),Org-20241 (Organon), Org-9731 (Organon), Zardaverine, vinpocetine, EHNA(MEP-1), Milrinone, Siguazodan, Zaprinast, SK+F 96231, Tolafentrine (BykGulden), Filaminast (Wyeth-Ayerst Pharmaceuticals).
 30. A method ofclaim 2 wherein the phosphodiesterase inhibitor is selected from thegroup consisting of: Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylic acid;3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide;2-(4- (6,7-Diethoxy-2,3-bis (hydroxymethyl) naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl) pthalazin-1 (2H)-one hydrochloride;7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.